10/7/13

Animal Experimental Study

Rovina Ruslami

Outline
introduction (definition, history)
why doing AES
how to do AES
objects
animal model
sample size

ethical consideration
some examples
where to publish
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INTRODUCTION
definition
history

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what is AES?...
the use of non-human animals in experiments

although some research about animals involve only pure observation/

natural behavior

pure research: genetics, developmental biology, behavioral studies

applied research (experiment): biomedical research,

xenotransplantation, drug development, cosmetic testing

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terminology
animal experimental study (AES)
animal experimentation
animal research
animal model
animal testing
in vivo testing
vivisection

drug development: preclinical trial

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history of AES
since 2nd 4th centuries BC
2nd-century: vivisection by Galen
12th-century: animal experimentation (surgery)
18th-century: discovery of drugs insulin, AB, vaccine
Laika (Sovyet dog); Dolly the sheep
20th-century: toxicology

1960s: Thalidomide safety testing on animal

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as usual

Debatepro- & anti-animal testing

increasing of AE on animal controversy

ethical issue
harm (for animal) vs. potential benefit (for human)
animal protection law

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WHY DOING AES?

for the science
for the benefit of humanity

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the need of
understanding of physiology, pathology, pathophysiology,
genetics of diseases
exploring and developing of new intervention (drugs, device,
etc.) of health problems
drugs:
ecacy (incl. pharmacokinetics, dose)
pharmacodynamics (mechanism of action)

testing (safety) before applying to human

drugs
safety
toxicology

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in general
1. Diseases process in human and animals have similarities
2. Cell systems contain or manipulate only a part of the
organ system.
3. Computer models lack the complexities of living entity

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...in drug development...

test of a new drug or a new medical device, done on
animal subjects, to see if the hoped-for treatment really
works and if it is safe to test on humans.
Its about ecacy (including pharmacodynamics: m.o.a)
and safety ( clinical trial)
before it goes into clinical trial (higher stage)

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AES in drug development

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HOW TO DO AES?
objects
animal model
sample size

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what animal?...
invertebrates vertebrates
non-human primates
50-100 million vertebrates/yr.

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invertebrates
fruit flies, worm
J short life cycle, ease with large numbers
L dierent immune system, simple organs
to identify active compound

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vertebrates
mice, rat, zebra fish, amphibians, cats, dogs, non-human primates

best model of inherited human disease

J genetic engineering model for a range of human disease

physiology, pharmacology, toxicology, cancer research, neurological

research, education J, endocrinology, reproduction, genetics,
HIV-AIDS drugs, vaccine, etc.

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source & fate

Source:
bred for a purpose lab, specialist suppliers (e.g. genetically
modified animal)
wild animal, bunching L
normal/healthy ones OR mutant
experiment could be:
usually one procedure
minutes, days, months or even years
fate: die because of
the experiment
euthanized after experiments
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Animal model
(of disease/condition)
to solve specific or practical problems
early stage of drug development

transgenic animal (inserted/modified/removed)

how and why disease develop?
test of new treatments

induced animal model

by virus, agent
inoculation of cancer cells

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Animal model: induction

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ETHICAL
CONSIDERATION

e 3-Rs

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Care and Use of animals

use non-animal methods over animal methods
Replacement (if possible)
use lower class of animal

use fewer animal for comparable levels of

information
Reduction use healthy animal, genetic homogeneity
obtain more information from the same no. of
animal

alleviate /minimize potential pain, suffering, or

distress
Refinement enhance animal welfare for their used
caring, treatment, non-invasive
(Russell & Burch, 1959)
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how to do 3-Rs?...

Replacement Reduction Refinement

cell culture
experimental less invasive
techniques techniques

better medical
computer models data analysis
care

human volunteers
sharing better living
information conditions

epidemiological
studies

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pain & suering

does animal feel pain suering???
testing causes distress & pain!
use of analgesia or anesthesia (local/general anesthesia)
what to use? what dose?
what if

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5-freedoms (Animal Welfare)

The council believes that the welfare of an animal ... should
be considered with reference to Five Freedoms.
v Freedom from hunger and thirst
v Freedom from discomfort
v Freedom from pain, injury and disease
v Freedom to express normal behaviour
v Freedom from fear and distress
(Animal Welfare Council UK, 1993)

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post-mortem examination OR euthanized how?

injection/inhalation

physical (decapitation/cervical dislocation) only after

maceration grinding into small pieces animal is
unconscious
irradiation of the brain (anesthetized)
captive bolts concussion of brain

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EXPERIMENTAL
DESIGN
Basic principles
Sample size calculation

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...basic principles...

remember, this an
experimental study
1. RQ
2. comparison/control
3. replication
4. randomization
5. stratification
6. factorial experiment

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...basic principles...

remember, this an 1. RQ: does salted drinking

experimental study water aect BP in mice?
1. RQ Experiment:
2. comparison/control
1. provide a mouse with water
3. replication
containing NaCl 1%
4. randomization
5. stratification 2. for 14 days
6. factorial experiment 3. measure BP

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2. Comparison/control:
good experiments are comparative
compare BP in mice fed with salt water to BP in mice fed plain water
compare BP in strain A mice fed salt water to BP in strain B mice fed
salt water
ideally they are compared to concurrent controls (rather than to
historical controls)

3. Replication
will increase precision decrease the sample size J

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4. Randomization:
to avoid bias and to control the chance
RAL J

5. Stratification
replication measure BP in the morning and some in the
afternoon (limited by personnel)
dierent of BP between morning & afternoon measurement?
ensure that within each period: equal number of objects in
each treatment group
latertake account of the di. between periods in stat. analysis

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example
20 M and 20 F mice randomized to be treated & untreated
can only work with 4 mice/day
Q: how to assign individuals to treatment group & to days?

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example
20 M and 20 F mice randomized to be treated & untreated
can only work with 4 mice/day
Q: how to assign individuals to treatment group & to days?

RANDOMIZED

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example
20 M and 20 F mice randomized to be treated & untreated
can only work with 4 mice/day
Q: how to assign individuals to treatment group & to days?

STRATIFIED DESIGN
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6. Factorial experiments:
to assess eect of > 1 treatment/intervention
exp.: eect of both salt water & high-fat diet on the BP (2
intervention
ideally: look at all 4 treatments in one experiments (&
interactions among them)
plain water normal diet
x
salt water high-fat diet
J we can learn more
more ecient
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in terms of sample size

Remember again:
too few animals a total waste! (no power to show the eect)
too many a partial waste

AND..3-Rs reduction
variability
reduce number of treatment group, if possible
more homogenous object (i.e. in breeding, control other
variables- BW, age, sex, etc. if possible)
use stratification
multiple measurement more precise the mean of measurement
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sample size: formula?

Federers formula
t: no. of treatment group (t.g)
(t-1) * (n-1) > 15 n: n/treatment group
exp. t=3 n/group > 8.5 = 9
N total = 27
Federer WT. Experimental design, theory and application, 1967

Meads equation
E: error D.o.F (10 20)
E=NBT

N: total samples needed
B: blocking/stratification
T: no. of treatment group

Festing MFW, et al. Reducing the animal in lab BMR, ATLA, 1998

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sample size: formula?

Meads equation
E: error D.o.F (10 20)
E=NBT N: total samples needed
B: blocking/stratification
T: no. of treatment group
Exp.
T = 3, B = 0 (10-20) =N-3
N = 13 23 5 7/group Federers formula
N total: 15 21 T = 3 (t 1)* (n 1) > 15
2 * (n 1) > 15
T (f.d) = 2x2x2 = 8 n 1 > 15/2 (= 7.5 = 8)
(10 20) = N (8) n > 9 N total: 27
N = 18 28 3/group
N total: 24 Meads equation
N total: 15 - 21
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TWO TYPES OF AES

Pharmacodynamics study
Toxicity study

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Pharmacodynamics study(1)
Route of administration:
Per Oral - similar to human use

Dose:
Based on Dose-Response Relationship
One or more doses that provide a desired eect
Dose conversion from human to animal may be used

Calculation of ED50

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Pharmacodynamics study(2)
Control group:
Negative control
Solvent / vehicle group

Positive control
Standard drug group
To validate that a method works
To obtain Relative Potency of drug candidate or herbal medicines

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Toxicological studies(1)
General toxicity test
Short-terms
Acute Toxicity Test (calculation of LD50)

Long-terms:
Sub Acute Test ( up to 1 month)
Sub Chronic Test ( up to 3 months)
Chronic Test ( up to 6 months)
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Toxicological studies(2)
Local toxicity test:
Dermatological Preparation

Special toxicity test:

Mutagenicity Test
Carcinogenicity Test
Reproductive& Development
Toxicity Test (teratogenicity)

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Animal model: infectious disease

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Animal model: infectious disease

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Animal model: infectious disease

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Animal model: metabolic disease

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like other studies/research..

methods are very crucial

as well as ethical aspects

validity of the data

statistical analysis

important path before next

step: clinical trial

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WHERE TO PUBLISH?

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Even the guidance

is there

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take home message

AES is a path of finding the answer for health problems
avoid doing AES only for a narrow purpose
just do it for the right reasons
animal are a living creature
do it correctly, appropriately the 3-Rs

thank you
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